11-oxygenated 9alpha-halogeno-1, 4-pregnadienes



United States Patent 3,084,103 II-OXYGENATED 9u-HALOGENO-L4-PREQNADIENES Arthur Nobiie, Livingston, NJ, assignor to SeheringJ:orporation, Bloomfield, NJL, a corporation of New ersey No Drawing.Filed Nov. 6, 1961, Ser. No. 150,160 23 Claims. (Cl. 167-74) Thisinvention relates to a new group of halogenated steroid dienes and toprocesses for their manufacture.

More particularly, this invention relates toll-oxygenated-9a-halogeno-1,4-pregnadiene-2l-ol-3,20 diones which may ormay not have a 17whydroxyl group, and their 21-carboxylic acid esters.

The present application is a continuation-in-part of my copendingapplications Serial No. 449,257, filed August 1'1, 1954, and Serial No.481,279, filed January 11, 1955, now United States Patent No. 2,837,464and Serial No. 5 13,902, filed June 7, 1955.

The =17-desoxy-l,4-pregnadiene compounds of this invention prepared fromthe corresponding 17-desoxy-4- pregnene compounds (described, forexample, in the patent to Fried, No. 2,852,511, dated September '16,1958), possess adrenocortical properties and are useful in the treatmentof Addisons disease and related diseases wherein such properties areindicated. The 17a-hydroxy compounds are generally useful for the samepurposes as 9u-halogeno-cortisone and hydrocortisone, but because oftheir enhanced activity can be employed in smaller dosages. The17-desoXy compounds can also be employed as intermediates for thepreparation of the corresponding l7a-hydroxyl compounds.

The novel compounds of this invention are compositions of matterrepresented by 9a-halogeno-3,ZO-diketo- 4-pregnenes having a hydroxy orester group at the 21- position, H or hydroxyl at the Um-position, ketoor 5- hydroxyl at the '1 l-position and characterized by the presence ofa double bond in the 1-position.

A more specific representation of my novel composition is depicted bythe following formula:

wherein X is a halogen having a lower atomic number than iodine, namelyfluorine, chlorine and bromine; Y is H or OH; R is a member of the groupconsisting of hydrogen and lower alkanoyl, aroyl, and other non-toxicacyl groups, like acetyl, propionyl, butyryl, succinyl, cyclohexylacetyl and propionyl, cyclopentyl acetyl and propionyl, dichloroacetyl,benzoyl, veratroyl, salicylyl, phthalyl, and the like; and R is a memberof the group consisting of keto and (H, BOH).

I have found that the introduction of an additional double bond into theA-ring of 9ca-halogeno-3,20-diketo- 4-pregnenes having a member of thegroup consisting of hydroxyl and acyloxy at the 2l-position, a member ofthe group consisting of H and OH at the l7a-position and a member of thegroup consisting of keto and (H, ,B-OH) at the ll-position affordscompounds which are more potent than the parent steroid. Thus, thecompounds of the present invention which are devoid of a 17a-hydroxylgroup (such absence being characteristic of mineralocorticalsubstances), surprisingly exhibit glucocorticoid activities. In additionto being potent glucocorticolds, they are effective in controllingelectrolyte balance as determined by liver glycogen assays and sodiumretention methods, respectively. The 17 a-hydroxy compounds of thepresent invention, on the other hand, exhibit strong anti-inflammatoryactivity. The novel products of this invention possess similarpharmacodynamic properties as do their corresponding 1,2-dehydroprecursor starting materials but to a considerably enhanced degree.Thus, the increased potency of the compounds of the invention over theparent mono-unsaturated steroid will permit the use of smaller dosesthan now employed, so that untoward side effects will be reduced or eveneliminated.

The compounds of the invention are prepared by subjecting thecorresponding mono-unsaturated steroid, or its 2l-ester, or itscorresponding 3-hydroxy compound or 3- ester (in which case, the doublebond will be between the C and C -carbons) to the action of adehydrogenating member of the family Corynebactriaceae. Especiallysatisfactory results have been obtained with species of the genusCorynebacterium, examples of which are Corynebacterium simplex (AmericanType Culture Collection No. 6946) and Corynebacterium hoagii (ATCC No.7055). The starting pregnene compound may also be subjected to theaction of the separated enzymes produced in a culture of the bacteria.Because the other genera of the family Corynebactriaceae, namelyListeria and Erysipelothrix, include pathogenic bacteria, the use ofmembers of the genus Corynebacterium is preferred, as many members ofsuch genus are non-pathogenic, including C. simplex and C. hoagii.

The cultures of the bacteria above referred to, or their enzymes, arecapable of dehydrogenating the starting compound at C and C therebyintroducing a double bond between such carbons. These cultures arecapable, however, of effecting additional chemical transformations:thus, an ester group at the 3-position will be hydrolyzed, while a3-hydroxyl group will be oxidized with simultane ous shifting of the5,6-double bond to the 4,5-position; and an ester group at the2l-position will generally also be hydrolyzed. The temperature ofincubation can range from about 25 C. or below to about 37 C., the lowertemperatures favoring hydrolysis of ester groups.

The obtained free-2l-ols may be esterified in the conventional manner,as by the action of an appropriate acid chloride or anhydride,preferably in the presence of pyridine. In general, the esters increasethe duration of activity of the corresponding alcohols.

The compounds of this invention may be administered orally,parenterally, or topically, as required. Thus, the compounds may beformulated into tablets, ointments and the like, by mixing with asubstantial quantity of a pharmaceutical carrier, such as gums,starches, sugars, and inert inorganic materials generally, in the caseof tablets, and with inert creamy or unguent materials in the case ofointments; while for parenteral administration, the compounds arepreferably prepared as aqueous or oil suspensions and administeredgenerally intramuscularly.

In order to obtain a desirable growth of, for example, Corynebacteriumsimplex (ATCC No. 6946) for the process of this invention, a suitablenutrient medium is prepared containing carbohydrate, organic nitrogen,cofactors, and inorganic salts. It is possible to omit the use ofcarbohydrate without completely impairing the growth of the organism.The steroid compound, in the solid condition or dissolved or suspendedin a water-miscible solvent which is non-toxic toward the organism, isadded to the cultivated microorganism in a broth medium under sterileconditions. This culture is then shaken, aerated or simultaneouslyaerated and agitated, in order to enhance the growth of themicroorganism and the biochemical conversion of the steroid substrate.The steriod maybe added to the broth medium and then inoculated with thebacterium, or the cultivated microorganism in broth medium may be addedto the steroid. In certain cases, depending on the condition of thereaction medium, it may be more desirable to obtain optimum growth ofthe microorganism before the addition of the steroid. Alternatively,enzyme preparations obtained in known manner from cultures of themicroorganism may be used in my process.

In carrying out my process, the bacterium, such as Corynebacteriumsimplex, is cultivated in a suitable nutrient medium under aerobicconditions. After cultivation of the microorganism, the cell mass may beharvested by centrifuging the nutrient broth, decanting the supernatantliquid and suspending the cell mass in saline. A suitable volume of thecell suspension is then seeded into a desirable nutrient medium forsupporting growth of the microorganism. The nutrient medium employed maybe a yeast extract (Difco), casein hydrolysate (N-Z-Amine) (Type B,Shetfield), corn steep liquor, water extract or soybean oil meal,lactalbumin hydrolysate (Edamine Sheflield enzymatic), fish solubles,and the like.

Inorganic salts ared esirable to maintain a pH level in the reactionmedium of between 6.8 and 7.2; but the use of such salts for bufferingthe reaction mixture may be omitted. The omission of inorganic saltscauses the pH to rise from an initial value of 6.8 to about 7.7-8. This,however, will still permit the formation of the desired steroidal endproducts. The optimum temperature for growth of the selectedmicroorganism is 37 C., but the temperatures may vary between 25 and 37,and even between and 40 C. The time of reaction may vary from as littleas 3 hours to as much as 48 hours. The length of time which is employedwill depend on the steroid which is being transformed. Anywater-miscible, non-toxic (to the organism) solvent may be employed todissolve or suspend the steroid prior to mixing with the culture. Iprefer to use ethanol or acetone in such amounts that the finalconcentration of these solvents in the reaction mixture is no higherthan about 7% and may amount to only traces; owing to evaporation, thefinal concentration of the organic solvent may even be practically zero.

Following the completion of the dehydrogenation process, which may beaccompanied by hydrolysis when 3-esters are used, with oxidation of the3-hydroxyl, the products of reaction may be recovered from the mixtureby extraction with a suitable water-immiscible solvent, by filtration,by adsorption on a suitable adsorbent, or by any of the other procedurescommonly used in the art. For extraction, chlorinated lowerhydrocarbons, ketones, and alcohols are useful. These includechloroform, methylene chloride, trichloroethane, ethylene dichloride,butanol, diethylketone, and others. I prefer to use extraction as themethod for isolating the steroidal products. Following extraction, theproducts may be isolated by concentration of the extracts to a smallvolume or to dryness. Purification of the residues may be thenaccomplished in several ways. In many instances, simplerecrystallizations from a, suitable solvent or solvent mixture, such asacetone, methylene chloride, ethanol, acetone-hexane, methylenechloride-hexane, etc. affords the desired dienone in excellent yield andhigh state of purity.

The following examples are illustrative of procedures for thepreparation of the compounds of this invention, but are not intended toindicate the scope thereof, such scope being defined in the appendedclaims.

EXAMPLE 1 1,4-Pregnadiene-9ot-Flu0r0-21-0l-3,11,20-Tri0ne To a 300 ml.Erlenmeyer flask are added 100 ml. of 0.1% yeast extract (Difco)containing 9.0 m1. of 0.2 M potassium dihydrogen phosphate and 9.0 ml.of 0.2 M disodium hydrogen phosphate. The flask and its contents aresterilized by autoclaving for 15 minutes at 120 C. and to the sterilemedium is added 1 ml. of a 1% suspension of Corynebaclerium simplex(ATCC No. 6946). The flask and its contents are incubated at 28 C. for24 hours.

To a second 300 ml. Erlenmeyer flask are added 2 ml. of ethanol and 25mg. of 9a-fluoro-ll-dehydrocorticosterone. The 24-hour growth culture istransferred asceptically to the flask containing the steroid and themixture is incubated at 28 C. and shaken for 10 hours. At the end ofthat time the reaction mixture is extracted thoroughly with chloroformand the chloroform extracts are concentrated to a residue. The residueis crystallized from acetone-hexane, affording 4 mg. of 9e-fluoro-1,4-pregnadiene-2 l-ol-3 ,l1,20-trione.

The 2l-acetate of the compound of this example is prepared by adding 0.3g. of acetic anhydride to a solution of l g. of the 21-01 in 20 ml. ofanhydrous pyridine. The reaction mixture is permitted to stand overnightand is then diluted with ice water. The resulting solid isrecrystallized from methylene chloride-hexane, affording crystalline9ot-fluoro-1,4-pregnadiene-2l-o1-3,l1,20-tri0ne 2l-acetate.

EXAMPLE 2 9a-Flu0r0-1,4-Pregnadiene-11fi,21-Di0l-3,20-Dion e Bysubstituting the starting steroid in Example 1 by9a-fluoro-4-pregnene-1113,2l-diol-3,20-dione, gram for gram, there areobtained 5 mg. of the compound of this example after recrystallizationfrom acetone-hexane.

The corresponding 2l-acetate of the compounds of this example can beobtained by acetylation with acetic anhydride in pyridine, as in Example1.

EXAMPLE 3 9cx-Chl0r0-1,4-Pregnadiene-11 5,21 -Di0l-3,20-Di0ne To a 300ml. Erlenmeyer flask are added ml. of 0.1% yeast extract (Difco)containing 9.0 ml. of 0.2 M potassium dihydrogen phosphate and 9.0 ml.of 0.2 M disodium hydrogen phosphate. The flask and its contents aresterilized by autoclaving for 15 minutes at C. and to the sterile mediumis added 1 ml. of a 1% suspension of Corynebacterium haagii (ATCC No.7055). The flask and contents are incubated at 28 C. for 24 hours.

To a second 300 ml. Erlenmeyer flask are added 2 ml. of ethanol and 25mg. of 9a-chlorocorticosterone. The 24-hour growth culture istransferred aseptically to the flask containing the steroid and themixture is incubated at 28 C. and shaken for two hours. At the end ofthat time the reaction mixture is extracted thoroughly with chloroformand the chloroform extracts are evaporated to a residue. The residue iscrystallized from acetone-hexane, affording 7 mg. of9a-chloro-1,4-pregnadiene-l1B,2l-diol-3,20-dione.

From the reaction of propionic anhydride and the diene obtained,according to the esterification procedure of Example 1, there isobtained Qua-chloro-1,4-pregnadiene- 1 1fl,21-3,20-dione 21-propionate.

EXAMPLE 4 9a-Bromo-1,4-Pregnadiene-11 8,21-dil-3,20-di0ne By employing9a-bromocorticosterone 21-acetate in the procedure described in Example3, the bromo-diene is obtained as a crystalline solid afterrecrystallization from acetone-hexane.

EXAMPLE 9a-Fluor0-1 ,4-Pregnadiene-1 113,1 7a,21-Tri0l-3,20-Di0ne A onehundred ml. broth culture containing a 0.1% yeast extract concentration,9.0 m1. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na HPO is seeded with 1 ml.of a 24-hour broth culture of Corynebacterium simplex. The flask isincubated at 28 C. for 24 hours. A second 300 ml. of Erlenmeyer flaskcontaining 150 mg. of sterile90cfluoro-4-pregnene-1118,17a,21-triol-3,20-dione in 5.0 ml. acetone isinoculated with the 24-hour culture of Corynebacterium simplex. Theculture-containing steroid solution is incubated for 48 hours at 28 to30 C.

The product is extracted with chloroform and isolated by evaporation todryness. Recrystallization of the residue afiords 9a-fluoro-A-pregnadiene-11,B,17a,2l-triol- 3,20-dione as a crystalline solid.

EXAMPLE 6 9a-Flu0r0-1 ,4-Pregnadiene-1 7a,21-Di0l-3,11,20-Tri0ne A brothculture is seeded as described in Example 5 and the reaction isperformed using 150 mg. of sterile9u-fluoro-4-pregnene-171:,21-diol-3,11,20-trione in 5.0 ml. of acetoneas reactant. Following the procedure of Example 5, the compound of thisexample is obtained as a crystalline solid.

EXAMPLE 7 By employing 9oz-Ch10I'0-4-p1'6g116116-170:,21-diO1-3,11,20-trione in the procedure described in Example 5, the 9mchloro-pregnadieneof this example is obtained in crystalline form.

EXAMPLE 8 9a-Br0mo-1,4-Pregnadiene-1 711,21-Diol-3,11,20-Tri0ne Bysubstituting the star-ting steroid in Example 1 by9a-bromo-4-pregnene-l7a,21-diol-3,11,20 trione, gram for gram, there areobtained 5 mg. of the bromo-pregnadiene of this example which ispurified by recrystallization from acetone-hexane.

EXAMPLE 9 9a-Clzl0r0-1,4-Pregnadiene-11,3,1 7a,21-Triol-3,20-Di0ne Byemploying 9a-chloro-4-pregnene-1113,17a,21-triol- 3,20-dione in theprocedure described in Example 5, the chloro-diene is obtained as acrystalline solid after recrystallization from acetone-hexane.

EXAMPLE 10 9a-Br0m0-1,4-Pregnadiene-11,8,17a,21-Tri0l-3,20-DioneUtilizing 9oz bromo-4-pregnene-11B,17a,21-triol-3,20- dione as thereacting steroid in the procedure of Example 5, the bromo-diene of thisexample is obtained as a crystalline solid after recrystallization fromacetone-hexane.

As above indicated, the 17-desoxy compounds of the present invention canserve as intermediates for the preparation of the corresponding17u-hydroxy compounds, i.e., the 9a-fiuoro, chloro and bromo derivativesof A -cortisone and A -hydrocortisone. The introduction of the17mhydroxy group can be effected by subjecting the 17- desoxy compoundsto the action of an hydroxylating organism chosen from the genusTrichothecium, such as T. roseum as described by Meystre et al.,Helvetica Chim. Acta: 37, 1548 (1954). Thus, 9u-fluoro-1,4-pregnadiene-21-ol-3,11,20-trione, dissolved in a small quantity of acetone, can be17u-hydroxy1ated in a culture of Trichothecium roseum in the mannerdescribed in the copending application of Hershel L. Herzog and EugeneP. Oliveto, Serial No. 484,588, filed January 27, 1955, now abandoned,to yield the 9a-flu01'0 derivative of A -cortisone. In similar fashion,9a-fluoro-1,4-pregnadiene-11,8,21-diol- 3,20-dione can be converted intothe corresponding chydroxy compound (the 9at-fiuoro derivative of A-hydrocortisone).

I claim:

1. A composition of matter selected from the group consisting ofl-l-keto and 11B-hydr0xy-9a-halogeno-A pregnenes having a keto group atthe 3- and 20-positions, a member of the group consisting of H and OH atthe 17a-position, a member of the group consisting of hydroxyl and loweraliphatic acyloxy at the 21-position and characterized by the presenceof a double bond in the 1- position.

2. A compound selected from pregnadienes having the formula:

CHrOR wherein X is a halogen atom having an atomic number lower thanthat of iodine, Y is a member of the group consisting of H and OH, R isa member of the group consisting of hydrogen and lower aliphatic acyl,and R is a member of the group consisting of 'keto and (H, fJ-OH).

3. 9a-halogeno 1,4 pregnadiene-2l-ol-3,11,20-trione, the halogen atomhaving an atomic number lower than that of iodine.

4. 9a-halogeno-1,4-pregnadiene-17a,21 diol 3,11,20- trione, the halogenatom having an atomic number lower than that of iodine.

5. 9a-halogeno-l,4-pregnadiene 116,21 diol 3,20- dione, the halogen atomhaving an atomic number lower than that of iodine.

6. 9a-halogeno-l,4-pregnadiene 11fl,17a,21-triol-3,20- dione, thehalogen atom having an atomic number lower than that of iodine.

7. 9a-fluoro-1,4-pregnadiene-21-ol-3,11,2 0-trione.

8. 9'a-fiuoro-1,4-pregnadiene-17a,21-diol-3,11,20-trione.

9. 9a-fiuoro 1,4 pregnadiene-21-0l-3,11,20-trione 21- acetate.

10. 9a-fiuoro 1,4 pregnadiene 1711,21 diol-3,11,20- trione 21-acetate.

11. 9a-fiuoro-1,4-pregnadiene-1 1fl,21-diol-3,20-dione.

12. 9a-fluoro-1,4-pregnadiene-11fl,17ot,2l -'triol 3,20- dione.

13. 9a-fiuoro-1,4-pregnadiene-115,21-diol 3,20 dione 21-acetate.

14. 9a-fluoro-1,4-pregnadiene 11,8,17a,21 diol 3,20- dione 21-acetate.

15. 9a-chlor0-1,4-pregnadiene-11fl,21-diol-3,20-dione.

16. 9a-chloro-1,4-pregnadiene 17a,21 diol 3,11,20- trione.

17. 9a-bromo-1,4-pregnadiene-1lfi,2l-diol-3,20-dione.

18. 9a-bromo-1,4-pregnadiene 1711,21 diol 3,11,20- trione.

19. A 3,11,20-triketo-9a-halo 1701,21 dihydroxy-A pregnadicne of theformula OHzOR oz ljorr x where X is a halogen atom having an atomicnumber lower than that of iodine and R is selected from the groupconsisting of H and lower hydrocarbon carbonyl.

20. A pharmaceutical preparation comprising 9a-fluorol-dehydrocortisoneand a pharmaceutical carrier.

21. A pharmaceutical preparation comprising 9a-fluoro- 1,4-pregnadiene-11B,17a,21-triol-3,20-dione 21-acetate and a pharmaceutical carrier.

22. A pharmaceutical preparation comprising a composition of matterselected from the group consisting of 11-keto and1lfi-hydroxy-9a-halogeno-A -pregnenes having a keto group at the 3-and20-positions, a member of the group consisting of H and OH at the170t-PDSltlOn, a member of the group consisting of hydroxyl and'lowerali- CHzOR wherein R is a member of the group consisting of hydrogen andalkanoyl, while X is a member of the group consisting of O and W is amember of the group consisting of fluorine, chlorine and bromine, and Yis a member of the group consisting of H and OH, mixed with a non-toxicpharmaceutical carrier.

No references cited.

23. A PHARMACEUTICAL COMPOSITION COMPRISING A COMPOUND OF THE FORMULA